Amperometric methods for the detection and quantification of constituents in biological samples are known. These methods comprise the following steps:
(1) contacting said constituent with an oxidase enzyme for which the constituent is a substrate, thereby producing a change in the concentration of an electroactive substance in said sample;
(2) contacting said sample containing said electroactive substance with an inert metal indicator electrode;
(3) measuring the current flow between said indicator electrode and a counter electrode at a preselected potential difference between said electrodes; and
(4) relating the current to the concentration of the constituent initially present in the biological sample.
A recognized problem associated with such methods is the fouling of the indicator (also referred to as sensor) electrode by macromolecules which are found in biological test samples. The art has seen the development of numerous designs to circumvent this problem.
U.S. Pat. No. 3,539,455 issued to Clark on Nov. 10, 1970, discloses an electrode system suitable for the amperometric (or polarographic) determination of simple sugars which are, themselves, not electrochemically active. In a preferred embodiment, a polarographically active anode (e.g., platinum) is separated from a test solution by a semi-permeable membrane through which glucose can pass but through which large molecules, especially enzymes, cannot pass. Between the membrane and the anode surface is an enzyme such as glucose oxidase. When the glucose diffuses through the membrane, it reacts with glucose oxidase to produce hydrogen peroxide which depolarizes the anode thereby generating a measurable current in associated electronics. The semi-permeable membrane not only serves to keep glucose oxidase from diffusing out into the test solution, but also serves to prevent the enzyme catalase (which catalyzes the breakdown of hydrogen peroxide into a nonelectroactive species) from diffusing into the enzyme-containing volume.
U.S. Pat. No. 3,912,614 issued to Sprachlen et al. on Oct. 14, 1975, discusses the use of platinum electrodes for amperometric measurements of constituents found in biological samples, as well as the limitations on the use of platinum for this purpose, e.g., poisoning. According to Sprachlen et al., the use of noble metal electrodes covered with membranes permeable to the constituent to be measured, such as those membranes taught by Clark, are not entirely satisfactory, partly because of the diffusion characteristics of the membrane materials relied upon by Clark and partly because of the lack of structural integrity of such materials when exposed to air, e.g., cracking and inability to rehydrate satisfactorily.
To circumvent these problems, Sprachlen et al. developed reversible membranes (ones which could be dried and rehydrated without loss of structural integrity). Sprachlen et al. departed from the thin films of the prior art and used, instead, thick films which were hemispheric in the area of diffusion. Sprachlen et al. used hydrophilic polymers in either thick or thin films although the former were preferred because of the surprising result that the diffusion rate of oxygen (a constituent of interest) increased as the thickness of the membrane increased--up to a point. When the thickness exceeded a certain value, the diffusion rate began to fall. Sprachlen et al. found that optimum membrane thickness was about 2 to 3.5 times that of the electrode diameter.
U.S. Pat. No. 4,041,933 issued to Reichenberger on Aug. 16, 1977, discloses a polarographic electrode for measuring constituents in physiological media. A noble metal cathode within an insulating sheath has one end exposed to the test solution. This end is covered with an oxygen permeable membrane, generally a hydrophilic material such as polymethacrylate, polystyrol or cellulose acetate.
Certain uses of perfluorosulfonic acid polymers in electrode systems have been described. Perfluorosulfonic acid polymers are available commercially from the Du Pont Company as the product sold under the trademark "Nafion".
U.S. Pat. No. 4,265,714 issued to Nolan et al. on May 5, 1981, discloses a gas detecting device utilizing a hydrated, solid polymer electrolyte ion transporting membrane in electrical contact with an improved catalytic graphite sensing electrode in conjunction with reference and counter electrodes. Among other things, the solid polymer electrolyte ion transporting membrane can be a hydrated copolymer of polytetrafluoroethylene and polysulfonyl fluoride vinyl ether containing pendant sulfonic acid groups.
Martin & Freiser, Anal. Chem., Volume 53, p 902 (1981), describe the use of perfluorosulfonate cation-exchange polymers in potentiometric, ion-selective electrodes.
Cajn et al., Anal. Chem., Volume 51, 1323 (1979), characterize previous thin layer electrode designs as expensive, difficult to construct and requiring unusual manipulative techniques. These workers describe a simple and inexpensive wire thin layer electrode and cell system for potentiometric measurements. The cell consists of an outside cylinder of Teflon.RTM. tubing containing a thin layer electrode assembly, a reference electrode and an auxiliary electrode. The thin layer electrode assembly consists of a gold wire surrounded by Nafion.RTM. membrane tubing. There is a space between the gold wire surface and the Nafion.RTM. membrane which serves as a thin layer cavity which contains the electrolysis solution. The reference electrode is also made with Nafion.RTM..
Rubinstein and Bard, J. Amer. Chem. Soc., Volume 103, 5007 (1981) describe electrodes made from Nafion.RTM. coated pyrolitic graphite, glassy carbon or platinum. The Nafion.RTM. was used as a matrix for the incorporation of an electroactive catalyst to form a chemically modified electrode surface. These investigators measured the chemiluminescence resulting from the interaction of the electroactive catalyst with oxalate ions in a test solution and demonstrated electrochemical regeneration of the catalyst.
From the review of the patents and literature references discussed above, it can be seen that many designs have been formulated to prevent fouling of indicator electrodes in the amperometric determination of constituents in biological samples. As some of these references themselves point out, many of these designs are difficult and expensive to construct or require careful adjustment of membrane thickness. Many have response times which are slower than that of a bare indicator electrode because of inherent design limitations on diffusion of electroactive species or constituent of interest to the electrode surface. Accordingly, there is a need for a simple, rapidly responsive, inexpensive indicator electrode useful for the amperometric determination of constituents in biological samples, particularly for constituents which are not electroactive but which are substrates for oxidase enzymes which can catalyze the breakdown of the constituent into an electroactive substance.